Influenza virus reassortment

ABSTRACT

New influenza donor strains for the production of reassortant influenza A viruses are provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a U.S. National Phase patent application ofPCT/EP2013/075294, filed Dec. 2, 2013, which claims priority to U.S.Provisional Application No. 61/732,809, filed Dec. 3, 2012, all of whichare herein incorporated by reference in the present disclosure in theirentirety.

This invention was made in part with Government support under grant no.HHSO10020100061C awarded by the Biomedical Advanced Research andDevelopment Authority (BARDA). The Government has certain rights in theinvention.

SUBMISSION OF SEQUENCE LISTING ON ASCII TEXT FILE

The content of the following submission on ASCII text file isincorporated herein by reference in its entirety: a computer readableform (CRF) of the Sequence Listing (file name: 529552005300SEQLIST.txt,date recorded: May 26, 2015, size: 107 KB).

TECHNICAL FIELD

This invention is in the field of influenza A virus reassortment.Furthermore, it relates to manufacturing vaccines for protecting againstinfluenza A viruses.

BACKGROUND ART

The most efficient protection against influenza infection is vaccinationagainst circulating strains and it is important to produce influenzaviruses for vaccine production as quickly as possible.

Wild-type influenza viruses often grow to low titres in eggs and cellculture. In order to obtain a better-growing virus strain for vaccineproduction it is currently common practice to reassort the circulatingvaccine strain with a faster-growing high-yield donor strain. This canbe achieved by co-infecting a culture host with the circulatinginfluenza strain (the vaccine strain) and the high-yield donor strainand selecting for reassortant viruses which contain the hemagglutinin(HA) and neuraminidase (NA) segments from the vaccine strain and theother viral segments (i.e. those encoding PB1, PB2, PA, NP, M₁, M₂, NS₁and NS₂) from the donor strain. Another approach is to reassort theinfluenza viruses by reverse genetics (see, for example references 1 and2).

Reference 3 reports that a reassortant influenza virus containing a PB1gene segment from A/Texas/1/77, the HA and NA segments from A/NewCaledonia/20/99, a modified PA segment derived from A/Puerto Rico/8/34and the remaining viral segments from A/Puerto Rico/8/34 shows increasedgrowth in cells.

There are currently only a limited number of donor strains forreassorting influenza viruses for vaccine manufacture, and the strainmost commonly used is the A/Puerto Rico/8/34 (A/PR/8/34) strain.However, reassortant influenza viruses comprising A/PR/8/34 backbonesegments do not always grow sufficiently well to ensure efficientvaccine manufacture. Thus, there is a need in the art to provide furtherand improved donor strains for influenza virus reassortment.

SUMMARY OF PREFERRED EMBODIMENTS

The inventors have now surprisingly discovered that influenza viruseswhich comprise backbone segments from two or more influenza donorstrains can grow faster in a culture host (particularly in cell culture)compared with reassortant influenza A viruses which contain all backbonesegments from the same donor strain. In particular, the inventors havefound that influenza viruses which comprise backbone segments derivedfrom two different high-yield donor strains can produce higher yieldreassortants with target vaccine-relevant HA/NA genes than reassortantsmade with either of the two original donor strains alone.

Reassortant influenza A viruses with backbone segments from two or moreinfluenza donor strains may comprise the HA segment and the PB1 segmentfrom different influenza A strains. In these reassortant influenzaviruses the PB1 segment is preferably from donor viruses with the sameinfluenza virus HA subtype as the vaccine strain. For example, the PB1segment and the HA segment may both be from influenza viruses with a H1subtype. The reassortant influenza A viruses may also comprise the HAsegment and the PB1 segment from different influenza A strains withdifferent influenza virus HA subtypes, wherein the PB1 segment is notfrom an influenza virus with a H3 HA subtype and/or wherein the HAsegment is not from an influenza virus with a H1 or H5 HA subtype. Forexample, the PB1 segment may be from a H1 virus and/or the HA segmentmay be from a H3 influenza virus.

The invention also provides reassortant influenza A viruses withbackbone segments from two or more influenza donor strains in which thePB1 segment is from the A/California/07/09 influenza strain. Thissegment may have at least 95% identity or 100% identity with thesequence of SEQ ID NO: 22. The reassortant influenza A virus may havethe H1 HA subtype. It will be understood that a reassortant influenzavirus according to this aspect of the invention will not comprise the HAand/or NA segments from A/California/07/09.

Where the reassortant influenza A virus comprises backbone segments fromtwo or three donor strains, each donor strain may provide more than oneof the backbone segments of the reassortant influenza A virus, but oneor two of the donor strains can also provide only a single backbonesegment.

Where the reassortant influenza A virus comprises backbone segments fromtwo, three, four or five donor strains, one or two of the donor strainsmay provide more than one of the backbone segments of the reassortantinfluenza A virus. In general the reassortant influenza A virus cannotcomprise more than six backbone segments. Accordingly, for example, ifone of the donor strains provides five of the viral segments, thereassortant influenza A virus can only comprise backbone segments from atotal of two different donor strains.

Where a reassortant influenza A virus comprises the PB1 segment fromA/Texas/1/77, it preferably does not comprise the PA, NP or M segmentfrom A/Puerto Rico/8/34. Where a reassortant influenza A virus comprisesthe PA, NP or M segment from A/Puerto Rico/8/34, it preferably does notcomprise the PB1 segment from A/Texas/1/77. In some embodiments, theinvention does not encompass reassortant influenza A viruses which havethe PB1 segment from A/Texas/1/77 and the PA, NP and M segments fromA/Puerto Rico/8/34. The PB1 segment from A/Texas/1/77 may have thesequence of SEQ ID NO: 27 and the PA, NP or M segments from A/PuertoRico/8/34 may have the sequence of SEQ ID NOs 28, 29 or 30,respectively.

Influenza A virus strains of the invention can grow to higher viraltitres in MDCK cells and/or in eggs in the same time and under the samegrowth conditions compared with reassortant influenza strains thatcomprise all backbone segments from the same influenza donor strain.

The invention also provides a reassortant influenza A virus comprisingat least one backbone viral segment from a donor strain, wherein thedonor strain is the A/California/07/09 influenza strain. When the atleast one backbone viral segment is the PA segment it may have asequence having at least 95% or at least 99% identity with the sequenceof SEQ ID NO: 15. When the at least one backbone viral segment is thePB1 segment, it may have a sequence having at least 95% or at least 99%identity with the sequence of SEQ ID NO: 16. When the at least onebackbone viral segment is the PB2 segment, it may have a sequence havingat least 95% or at least 99% identity with the sequence of SEQ ID NO:17. When the at least one backbone viral segment is the NP segment itmay have a sequence having at least 95% or at least 99% identity withthe sequence of SEQ ID NO: 18. When the at least one backbone viralsegment is the M segment it may have a sequence having at least 95% orat least 99% identity with the sequence of SEQ ID NO: 19. When the atleast one backbone viral segment is the NS segment it may have asequence having at least 95% or at least 99% identity with the sequenceof SEQ ID NO: 20.

At least one backbone segment may be derived from the A/California/07/09influenza strain, as discussed in the previous paragraph. Preferredreassortant influenza A viruses comprise the PB1 segment from theA/California/07/09 influenza strain. The inventors have shown thatreassortant influenza A viruses comprising this backbone segment growwell in culture hosts. The reassortant influenza A viruses may compriseall other backbone segments from an influenza virus which is notA/California/07/09.

The reassortant influenza A viruses may comprise the PB1 segment fromA/California/07/09 and all other backbone segments from the influenzastrain PR8-X. The segments of PR8-X have the sequences of SEQ ID NO: 1(PA), SEQ ID NO: 2 (PB1), SEQ ID NO: 3 (PB2), SEQ ID NO: 4 (NP), SEQ IDNO: 5 (M), SEQ ID NO: 6 (NS), SEQ ID NO: 7 (HA) or SEQ ID NO: 8 (NA).Thus, the influenza viruses of the invention may comprise one or moregenome segments selected from: a PA segment having at least 95% or 99%identity to the sequence of SEQ ID NO: 1, a PB2 segment having at least95% or 99% identity to the sequence of SEQ ID NO: 3, a M segment havingat least 95% or 99% identity to the sequence of SEQ ID NO: 5, a NPsegment having at least 95% or 99% identity to the sequence of SEQ IDNO: 4, and/or a NS segment having at least 95% or 99% identity to thesequence of SEQ ID NO: 6. The reassortant influenza A viruses may alsocomprise one or more viral segments which have the sequence of SEQ IDNOs: 1, and/or 3-6. In preferred embodiments, the reassortant influenzastrain comprises all of the genome segments mentioned in this paragraph.This embodiment is preferred because the inventors have found that suchreassortant influenza A viruses grow particularly well in cell cultureand in embryonated hens eggs.

In general a reassortant influenza virus will contain only one of eachbackbone segment. For example, when the influenza virus comprises thePB1 segment from A/California/07/09 it will not at the same timecomprise the PB1 segment from another influenza A donor strain.

The backbone viral segments may be optimized for culture in the specificculture host. For example, where the reassortant influenza viruses arecultured in mammalian cells, it is advantageous to adapt at least one ofthe viral segments for optimal growth in the culture host. For example,where the expression host is a canine cell, such as a MDCK cell line,the viral segments may have a sequence which optimises viral growth inthe cell. Thus, the reassortant influenza viruses of the invention maycomprise a PB2 genome segment which has lysine in the positioncorresponding to amino acid 389 of SEQ ID NO: 3 when aligned to SEQ IDNO: 3 using a painvise alignment algorithm, and/or asparagine in theposition corresponding to amino acid 559 of SEQ ID NO: 3 when aligned toSEQ ID NO: 3 using a pairwise alignment algorithm. Also provided arereassortant influenza viruses in accordance with the invention in whichthe PA genome segment has lysine in the position corresponding to aminoacid 327 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1 using a pairwisealignment algorithm, and/or aspartic acid in the position correspondingto amino acid 444 of SEQ ID NO: 1 when aligned to SEQ ID NO: 1, using apairwise alignment algorithm, and/or aspartic acid in the positioncorresponding to amino acid 675 of SEQ ID NO: 1 when aligned to SEQ IDNO: 1, using a pairwise alignment algorithm. The reassortant influenzastrains of the invention may also have a NP genome segment withthreonine in the position corresponding to amino acid 27 of SEQ ID NO: 4when aligned to SEQ ID NO: 4 using a pairwise alignment algorithm,and/or asparagine in the position corresponding to amino acid 375 of SEQID NO: 4 when aligned to SEQ ID NO: 4, using a pairwise alignmentalgorithm. Variant influenza strains may also comprise two or more ofthese mutations. It is preferred that the variant influenza viruscontains a variant PB2 segment with both of the amino acids changesidentified above, and/or a PA which contains all three of the amino acidchanges identified above, and/or a NP segment which contains both of theamino acid changes identified above. The influenza A virus may be a H1strain.

Alternatively, or in addition, the reassortants influenza viruses maycomprise a PB1 segment which has isoleucine in the positioncorresponding to amino acid 200 of SEQ ID NO: 2 when aligned to SEQ IDNO: 2 using a pairwise alignment algorithm, and/or asparagine in theposition corresponding to amino acid 338 of SEQ ID NO: 2 when aligned toSEQ ID NO: 2 using a pairwise alignment algorithm, and/or isoleucine inthe position corresponding to amino acid 529 of SEQ ID NO: 2 whenaligned to SEQ ID NO: 2 using a pairwise alignment algorithm, and/orisoleucine in the position corresponding to amino acid 591 of SEQ ID NO:2 when aligned to SEQ ID NO: 2 using a pairwise alignment algorithm,and/or histidine in the position corresponding to amino acid 687 of SEQID NO: 2 when aligned to SEQ ID NO: 2 using a pairwise alignmentalgorithm, and/or lysine in the position corresponding to amino acid 754of SEQ ID NO: 2 when aligned to SEQ ID NO: 2 using a pairwise alignmentalgorithm.

The preferred pairwise alignment algorithm is the Needleman-Wunschglobal alignment algorithm [4], using default parameters (e.g. with Gapopening penalty=10.0, and with Gap extension penalty=0.5, using theEBLOSUM62 scoring matrix). This algorithm is conveniently implemented inthe needle tool in the EMBOSS package [5].

The invention provides a method of preparing the reassortant influenza Aviruses of the invention. These methods comprise steps of (i)introducing into a culture host one or more expression construct(s)which encode(s) the viral segments required to produce an influenza Avirus wherein the backbone viral segments are from two or more influenzastrains; and (ii) culturing the culture host in order to producereassortant virus and optionally (iii) purifying the virus obtained instep (ii). In these methods, the HA and the PB1 segment may be fromdifferent influenza strains which have the same influenza HA subtype orthe HA and PB1 segments may be from different influenza strains withdifferent HA subtypes provided that the PB1 segment is not from aninfluenza virus with a H3 HA subtype and/or the HA segment is not froman influenza virus with a H1 or H5 HA subtype. The PB1 backbone viralsegment may be from A/California/07/09. The one or more expressionconstructs may further encode one or more of the PB2, PA, NP, M, or NSsegments from PR8-X or segments having at least 90% or 100% identity toSEQ ID NOs: 9, and/or 11 to 14. The expression construct(s) may notencode the HA and/or NA segments from A/California/07/09 when the PB1segment is from A/California/07/09.

The at least one expression construct may comprise a sequence having atleast 90%, at least 95%, at least 99% or 100% identity with the sequenceof SEQ ID NO: 22.

In some embodiments, the at least one expression construct does notencode the PB1 segment from the A/Texas/1/77 influenza strain.

The methods may further comprise steps of: (iv) infecting a culture hostwith the virus obtained in step (ii) or step (iii); (v) culturing theculture host from step (iv) to produce further virus; and optionally(vi) purifying the virus obtained in step (v).

The invention also provides a method for producing influenza virusescomprising steps of (a) infecting a culture host with a reassortantvirus of the invention; (b) culturing the host from step (a) to producethe virus; and optionally (c) purifying the virus obtained in step (b).

The invention also provides a method of preparing a vaccine, comprisingsteps of (d) preparing a virus by the methods of any one of theembodiments described above and (e) preparing vaccine from the virus.

The invention provides an expression system comprising one or moreexpression construct(s) comprising the vRNA encoding segments of aninfluenza A virus wherein the expression construct(s) encode(s) the HAand PB1 segments from two different influenza strains with the sameinfluenza HA subtype or which encodes the HA and PB1 segments from twodifferent influenza strains with different influenza virus HA subtypes,wherein the PB1 segment is not from an influenza virus with a H3 HAsubtype and/or the HA segment is not from an influenza virus with a H1or H5 HA subtype.

The invention also provides an expression system comprising one or moreexpression construct(s) comprising the vRNA encoding segments of aninfluenza A virus wherein the expression construct(s) encode(s) the PB1segment of A/California/07/09. The expression construct(s) may furthercomprise the vRNAs which encode one or more of the PB2, NP, NS, M and/orPA segments from PR8-X. Thus, the expression construct(s) may compriseone or more nucleotide sequences having at least 90% identity, at least95% identity, at least 99% identity or 100% identity with the sequencesof SEQ ID NOs: 9 and/or 11-14. It is preferred that the expressionconstruct(s) encode(s) all of the PB2, NP, NS, M and PA segments fromPR8-X.

The invention also provides a host cell comprising the expressionsystems of the invention. These host cells can express an influenza Avirus from the expression construct(s) in the expression system.

Expression constructs which can be used in the expression systems of theinvention are also provided. For example, the invention provides anexpression construct which encodes the backbone segments of thereassortant influenza strains according to the invention on the sameconstruct.

Donor Strains

Influenza donor strains are strains which typically provide the backbonesegments in a reassortant influenza virus, even though they maysometimes also provide the NA segment of the virus. Usually, however,both the HA and the NA segment in a reassortant influenza virus will befrom the vaccine strain which is the influenza strain that provides theHA segment.

The inventors have surprisingly discovered that reassortant influenza Aviruses which comprise the HA segment and the PB1 segment from differentinfluenza A strains with the same HA subtype can grow much faster inculture hosts compared with reassortant influenza viruses which comprisethe HA and PB1 segments from viruses with different HA subtypes. Thesereassortant influenza viruses preferably have backbone segments from atleast two donor strains.

The PB1 segments of influenza viruses with the same HA subtype willusually have a higher level of identity than the PB1 segments ofinfluenza viruses with different HA subtypes. For example, a Blastsearch using the PB1 segment of the H1 strain A/California/07/09 showedthat only influenza strains with the H1 HA subtype had a high identityin the PB1 segment. Likewise, a Blast search using the PB1 segment ofthe H3 strain A/Wisconsin/67/2005 showed that only influenza viruseswith the H3 HA subtype had a high level of identity to the PB1 segmentof this virus.

The inventors have further discovered that reassortant influenza Aviruses which have backbone segments from at least two donor strains andcomprise the PB1 segment from A/California/07/09 grow particularly wellin culture hosts. These reassortant influenza viruses preferably havebackbone segments from at least two different donor strains. Thereassortant influenza viruses may comprise the PB1 segment fromA/California/07/09 and the HA segment of an influenza virus with the H1subtype.

Influenza strains which contain one, two, three, four five, six or sevenof the segments of the A/California/07/09 strain can also be used asdonor strains.

The invention can be practised with donor strains having a viral segmentthat has at least about 70%, at least about 75%, at least about 80%, atleast about 85%, at least about 90%, at least about 95% or at leastabout 99% identity to a sequence of SEQ ID NOs 9-14 or 21-26. Forexample, due to the degeneracy of the genetic code, it is possible tohave the same polypeptide encoded by several nucleic acids withdifferent sequences. Thus, the invention may be practised with viralsegments that encode the same polypeptides as the sequences of SEQ IDNOs 1-8 or 15-20. For example, the nucleic acid sequences of SEQ ID NOs:31 and 32 have only 73% identity even though they encode the same viralprotein.

The invention may also be practised with viral segments that encodepolypeptides that have at least 80%, at least 85%, at least 90%, atleast 95% or at least 99% identity to the polypeptide sequences encodedby SEQ ID NOs 9-22.

Variations in the DNA and the amino acid sequence may also stem fromspontaneous mutations which can occur during passaging of the viruses.Such variant influenza strains can also be used in the invention.

Reassortant Viruses

The invention provides reassortant influenza viruses which comprisebackbone segments from two or more influenza donor strains. Thesereassortant influenza viruses may comprise the HA segment and the PB1segment from different influenza A strains provided that the HA and thePB1 segments are from influenza viruses with the same influenza virus HAsubtype. They may also comprise the HA segment and the PB1 segment fromdifferent influenza A strains with different influenza virus HAsubtypes, provided that the PB1 segment is not from an influenza viruswith a H3 HA subtype and/or the HA segment is not from an influenzavirus with a H1 or H5 HA subtype.

Further provided are reassortant influenza viruses with backbonesegments from two or more different donor strains which comprise the PB1segment from A/California/07/09.

The PB1 and PB2 segments may be from the same donor strain.

Influenza viruses are segmented negative strand RNA viruses. Influenza Aand B viruses have eight segments (NP, M, NS, PA, PB1, HA and NA)whereas influenza C virus has seven. The reassortant viruses of theinvention contain the backbone segments from two or more donor strains,or at least one (i.e. one, two, three, four, five or six) backbone viralsegment from A/California/07/09. The backbone viral segments are thosewhich do not encode HA or NA. Thus, backbone segments will typicallyencode the PB1, PB2, PA, NP, M₁, M₂, NS₁ and NS₂ polypeptides of theinfluenza virus. The viruses may also contain an NS segment that doesnot encode a functional NS protein as described, for example, inreference 6. The reassortant viruses will not typically contain thesegments encoding HA and NA from the donor strains even thoughreassortant viruses which comprise either the HA or the NA but not bothfrom the donor strains of the invention are also envisioned.

When the reassortant viruses are reassortants comprising the backbonesegments from a single donor strain, the reassortant viruses willgenerally include segments from the donor strain and the vaccine strainin a ratio of 1:7, 2:6, 3:5, 4:4, 5:3, 6:2 or 7:1. Having a majority ofsegments from the donor strain, in particular a ratio of 6:2, istypical. When the reassortant viruses comprise backbone segments fromtwo donor strains, the reassortant virus will generally include segmentsfrom the first donor strain, the second donor strain and the vaccinestrain in a ratio of 1:1:6, 1:2:5, 1:3:4, 1:4:3, 1:5:2, 1:6:1, 2:1:5,2:2:4, 2:3:3, 2:4:2, 2:5:1, 3:1:2, 3:2:1, 4:1:3, 4:2:2, 4:3:1, 5:1:2,5:2:1 or 6:1:1.

Preferably, the reassortant viruses do not contain the HA segment of thedonor strain as this encodes the main vaccine antigens of the influenzavirus and should therefore come from the vaccine strain. The reassortantviruses of the invention therefore preferably have at least the HAsegment and typically the HA and NA segments from the vaccine strain.

The invention also encompasses reassortants which comprise viralsegments from more than one vaccine strain provided that the reassortantcomprises a backbone according to the present invention. For example,the reassortant influenza viruses may comprise the HA segment from onedonor strain and the NA segment from a different donor strain.

The reassortant viruses of the invention can grow to higher viral titresthan the wild-type vaccine strain from which some of the viralsegment(s) of the reassortant virus are derived in the same time (forexample 12 hours, 24 hours, 48 hours or 72 hours) and under the samegrowth conditions. The viral titre can be determined by standard methodsknown to those of skill in the art. The reassortant viruses of theinvention can achieve a viral titre which is at least 10% higher, atleast 20% higher, at least 50% higher, at least 100% higher, at least200% higher, at least 500% higher, or at least 1000% higher than theviral titre of the wild type vaccine strain in the same time frame andunder the same conditions.

The invention is suitable for reassorting pandemic as well asinter-pandemic (seasonal) influenza vaccine strains. The reassortantinfluenza strains may contain the influenza A virus HA subtypes H1, H2,H3, H4, H5, H6, H7, H8, H9, H10, H11, H12, H13, H14, H15 or H16. Theymay contain the influenza A virus NA subtypes N1, N2, N3, N4, N5, N6,N7, N8 or N9. Where the vaccine strain used in the reassortant influenzaviruses of the invention is a seasonal influenza strain, the vaccinestrain may have a H1 or H3 subtype. In one aspect of the invention thevaccine strain is a H1N1 or H3N2 strain. The reassortants influenzastrains may also contain the HA segment of an influenza B strain.

The vaccine strains for use in the invention may also be pandemicstrains or potentially pandemic strains. The characteristics of aninfluenza strain that give it the potential to cause a pandemic outbreakare: (a) it contains a new hemagglutinin compared to the hemagglutininsin currently-circulating human strains, i.e. one that has not beenevident in the human population for over a decade (e.g. H2), or has notpreviously been seen at all in the human population (e.g. H5, H6 or H9,that have generally been found only in bird populations), such that thehuman population will be immunologically naïve to the strain'shemagglutinin; (b) it is capable of being transmitted horizontally inthe human population; and (c) it is pathogenic to humans. A vaccinestrain with a H5 hemagglutinin type is preferred where the reassortantvirus is used in vaccines for immunizing against pandemic influenza,such as a H5N1 strain. Other possible strains include H5N3, H9N2, H2N2,H7N1 and H7N7, and any other emerging potentially pandemic strains. Theinvention is particularly suitable for producing reassortant viruses foruse in a vaccine for protecting against potential pandemic virus strainsthat can or have spread from a non-human animal population to humans,for example a swine-origin H1N1 influenza strain.

The reassortant influenza strain of the invention may comprise the HAsegment and/or the NA segment from an A/California/4/09 strain.

Strains which can be used as vaccine strains include strains which areresistant to antiviral therapy (e.g. resistant to oseltamivir [7] and/orzanamivir), including resistant pandemic strains [8].

Reassortant viruses which contain an NS segment that does not encode afunctional NS protein are also within the scope of the presentinvention. NS1 knockout mutants are described in reference 6. TheseNS1-mutant virus strains are particularly suitable for preparing liveattenuated influenza vaccines.

The ‘second influenza strain’ used in the methods of the invention isdifferent to the donor strain which is used.

Reverse Genetics

The invention is particularly suitable for producing the reassortantinfluenza virus strains of the invention through reverse geneticstechniques. In these techniques, the viruses are produced in culturehosts using an expression system.

In one aspect, the expression system may encode the HA and PB1 segmentfrom different influenza strains with the same HA subtype. It may alsoencode the HA and PB1 segments from different influenza strains withdifferent HA subtypes provided that the PB1 segment is not from aninfluenza virus with a H3 HA subtype and/or the HA segment is not froman influenza virus with a H1 or H5 HA subtype. The expression system mayencode the PB1 segment from A/California/07/09. In these embodiments,the system may encode at least one of the segments NP, M, NS, PA, and/orPB2 from another influenza donor strain, for example PR8-X.

Reverse genetics for influenza A and B viruses can be practised with 12plasmids to express the four proteins required to initiate replicationand transcription (PB1, PB2, PA and nucleoprotein) and all eight viralgenome segments. To reduce the number of constructs, however, aplurality of RNA polymerase I transcription cassettes (for viral RNAsynthesis) can be included on a single plasmid (e.g. sequences encoding1, 2, 3, 4, 5, 6, 7 or all 8 influenza vRNA segments), and a pluralityof protein-coding regions with RNA polymerase II promoters on anotherplasmid (e.g. sequences encoding 1, 2, 3, 4, 5, 6, 7 or 8 influenza mRNAtranscripts) [9]. It is also possible to include one or more influenzavRNA segments under control of a pol I promoter and one or moreinfluenza protein coding regions under control of another promoter, inparticular a pol II promoter, on the same plasmid. This is preferablydone by using bi-directional plasmids.

Preferred aspects of the reference 9 method involve: (a) PB1, PB2 and PAmRNA-encoding regions on a single expression construct; and (b) all 8vRNA encoding segments on a single expression construct. Including theneuraminidase (NA) and hemagglutinin (HA) segments on one expressionconstruct and the six other viral segments on another expressionconstruct is particularly preferred as newly emerging influenza virusstrains usually have mutations in the NA and/or HA segments. Therefore,the advantage of having the HA and/or NA segments on a separateexpression construct is that only the vector comprising the HA and NAsequence needs to be replaced. Thus, in one aspect of the invention theNA and/or HA segments of the vaccine strain may be included on oneexpression construct and the vRNA encoding segments from the donorstrain(s) of the invention, excluding the HA and/or NA segment(s), areincluded on a different expression construct. The invention thusprovides an expression construct comprising one, two, three, four, fiveor six vRNA encoding backbone viral segments of a donor strain of theinvention. The expression construct may not comprise HA and/or NA viralsegments that produce a functional HA and/or NA protein.

Known reverse genetics systems involve expressing DNA molecules whichencode desired viral RNA (vRNA) molecules from pol I promoters,bacterial RNA polymerase promoters, bacteriophage polymerase promoters,etc. As influenza viruses require the presence of viral polymerase toinitiate the life cycle, systems may also provide these proteins e.g.the system further comprises DNA molecules that encode viral polymeraseproteins such that expression of both types of DNA leads to assembly ofa complete infectious virus. It is also possible to supply the viralpolymerase as a protein.

Where reverse genetics is used for the expression of influenza vRNA, itwill be evident to the person skilled in the art that precise spacing ofthe sequence elements with reference to each other is important for thepolymerase to initiate replication. It is therefore important that theDNA molecule encoding the viral RNA is positioned correctly between thepol I promoter and the termination sequence, but this positioning iswell within the capabilities of those who work with reverse geneticssystems.

In order to produce a recombinant virus, a cell must express allsegments of the viral genome which are necessary to assemble a virion.DNA cloned into the expression constructs of the present inventionpreferably provides all of the viral RNA and proteins, but it is alsopossible to use a helper virus to provide some of the RNA and proteins,although systems which do not use a helper virus are preferred. As theinfluenza virus is a segmented virus, the viral genome will usually beexpressed using more than one expression construct in the methods of theinvention. It is also envisioned, however, to combine one or moresegments or even all segments of the viral genome on a single expressionconstruct.

In some embodiments an expression construct will also be included whichleads to expression of an accessory protein in the host cell. Forinstance, it can be advantageous to express a non-viral serine protease(e.g. trypsin) as part of a reverse genetics system.

Expression Constructs

Expression constructs used in the expression systems of the inventionmay be uni-directional or bi-directional expression constructs. Wheremore than one transgene is used in the methods (whether on the same ordifferent expression constructs) it is possible to use uni-directionaland/or bi-directional expression.

As influenza viruses require a protein for infectivity, it is generallypreferred to use bi-directional expression constructs as this reducesthe total number of expression constructs required by the host cell.Thus, the method of the invention may utilise at least onebi-directional expression construct wherein a gene or cDNA is locatedbetween an upstream pol II promoter and a downstream non-endogenous polI promoter. Transcription of the gene or cDNA from the pol II promoterproduces capped positive-sense viral mRNA which can be translated into aprotein, while transcription from the non-endogenous pol I promoterproduces negative-sense vRNA. The bi-directional expression constructmay be a bi-directional expression vector.

Bi-directional expression constructs contain at least two promoterswhich drive expression in different directions (i.e. both 5′ to 3′ and3′ to 5′) from the same construct. The two promoters can be operablylinked to different strands of the same double stranded DNA. Preferably,one of the promoters is a pol I promoter and at least one of the otherpromoters is a pol II promoter. This is useful as the pol I promoter canbe used to express uncapped vRNAs while the pol II promoter can be usedto transcribe mRNAs which can subsequently be translated into proteins,thus allowing simultaneous expression of RNA and protein from the sameconstruct. Where more than one expression construct is used within anexpression system, the promoters may be a mixture of endogenous andnon-endogenous promoters.

The pol I and pol II promoters used in the expression constructs may beendogenous to an organism from the same taxonomic order from which thehost cell is derived. Alternatively, the promoters can be derived froman organism in a different taxonomic order than the host cell. The term“order” refers to conventional taxonomic ranking, and examples of ordersare primates, rodentia, carnivora, marsupialia, cetacean, etc. Humansand chimpanzees are in the same taxonomic order (primates), but humansand dogs are in different orders (primates vs. carnivora). For example,the human pol I promoter can be used to express viral segments in caninecells (e.g. MDCK cells) [10].

The expression construct will typically include an RNA transcriptiontermination sequence. The termination sequence may be an endogenoustermination sequence or a termination sequence which is not endogenousto the host cell. Suitable termination sequences will be evident tothose of skill in the art and include, but are not limited to, RNApolymerase I transcription termination sequence, RNA polymerase IItranscription termination sequence, and ribozymes. Furthermore, theexpression constructs may contain one or more polyadenylation signalsfor mRNAs, particularly at the end of a gene whose expression iscontrolled by a pol II promoter.

An expression system may contain at least two, at least three, at leastfour, at least five, at least six, at least seven, at least eight, atleast nine, at least ten, at least eleven or at least twelve expressionconstructs.

An expression construct may be a vector, such as a plasmid or otherepisomal construct. Such vectors will typically comprise at least onebacterial and/or eukaryotic origin of replication. Furthermore, thevector may comprise a selectable marker which allows for selection inprokaryotic and/or eukaryotic cells. Examples of such selectable markersare genes conferring resistance to antibiotics, such as ampicillin orkanamycin. The vector may further comprise one or more multiple cloningsites to facilitate cloning of a DNA sequence.

As an alternative, an expression construct may be a linear expressionconstruct. Such linear expression constructs will typically not containany amplification and/or selection sequences. However, linear constructscomprising such amplification and/or selection sequences are also withinthe scope of the present invention. Reference 11 describes a linearexpression construct which describes individual linear expressionconstructs for each viral segment. It is also possible to include morethan one, for example two, three four, five or six viral segments on thesame linear expression construct. Such a system has been described, forexample, in reference 12.

Expression constructs can be generated using methods known in the art.Such methods were described, for example, in reference 13. Where theexpression construct is a linear expression construct, it is possible tolinearise it before introduction into the host cell utilising a singlerestriction enzyme site. Alternatively, it is possible to excise theexpression construct from a vector using at least two restriction enzymesites. Furthermore, it is also possible to obtain a linear expressionconstruct by amplifying it using a nucleic acid amplification technique(e.g. by PCR).

The expression constructs used in the systems of the invention may benon-bacterial expression constructs. This means that the construct candrive expression in a eukaryotic cell of viral RNA segments encodedtherein, but it does not include components which would be required forpropagation of the construct in bacteria. Thus the construct will notinclude a bacterial origin of replication (ori), and usually will notinclude a bacterial selection marker (e.g. an antibiotic resistancemarker). Such expression constructs are described in reference 14 whichis incorporated by reference.

The expression constructs may be prepared by chemical synthesis. Theexpression constructs may either be prepared entirely by chemicalsynthesis or in part. Suitable methods for preparing expressionconstructs by chemical synthesis are described, for example, inreference 14 which is incorporated by reference.

The expression constructs of the invention can be introduced into hostcells using any technique known to those of skill in the art. Forexample, expression constructs of the invention can be introduced intohost cells by employing electroporation, DEAE-dextran, calcium phosphateprecipitation, liposomes, microinjection, or microparticle-bombardment.

Cells

The culture host for use in the present invention can be any eukaryoticcell that can produce the virus of interest. The invention willtypically use a cell line although, for example, primary cells may beused as an alternative. The cell will typically be mammalian or avian.Suitable mammalian cells include, but are not limited to, hamster,cattle, primate (including humans and monkeys) and dog cells. Variouscell types may be used, such as kidney cells, fibroblasts, retinalcells, lung cells, etc. Examples of suitable hamster cells are the celllines having the names BHK21 or HKCC. Suitable monkey cells are e.g.African green monkey cells, such as kidney cells as in the Vero cellline [15-17]. Suitable dog cells are e.g. kidney cells, as in the CLDKand MDCK cell lines.

Further suitable cells include, but are not limited to: CHO; 293T; BHK;MRC 5; PER.C6 [18]; FRhL2; WI-38; etc. Suitable cells are widelyavailable e.g. from the American Type Cell Culture (ATCC) collection[19], from the Coriell Cell Repositories [20], or from the EuropeanCollection of Cell Cultures (ECACC). For example, the ATCC suppliesvarious different Vero cells under catalogue numbers CCL 81, CCL 81.2,CRL 1586 and CRL-1587, and it supplies MDCK cells under catalogue numberCCL 34. PER.C6 is available from the ECACC under deposit number96022940.

Preferred cells for use in the invention are MDCK cells [21-23], derivedfrom Madin Darby canine kidney. The original MDCK cells are availablefrom the ATCC as CCL 34. It is preferred that derivatives of MDCK cellsare used. Such derivatives were described, for instance, in reference 21which discloses MDCK cells that were adapted for growth in suspensionculture (‘MDCK 33016’ or ‘33016-PF’, deposited as DSM ACC 2219; see alsoref. 21). Furthermore, reference 24 discloses MDCK-derived cells thatgrow in suspension in serum free culture (‘B-702’, deposited as FERMBP-7449). In some embodiments, the MDCK cell line used may betumorigenic. It is also envisioned to use non-tumorigenic MDCK cells.For example, reference 25 discloses non tumorigenic MDCK cells,including ‘MDCK-S’ (ATCC PTA-6500), ‘MDCK-SF101’ (ATCC PTA-6501),‘MDCK-SF102’ (ATCC PTA-6502) and ‘MDCK-SF103’ (ATCC PTA-6503). Reference26 discloses MDCK cells with high susceptibility to infection, including‘MDCK.5F1’ cells (ATCC CRL 12042).

It is possible to use a mixture of more than one cell type to practisethe methods of the present invention. However, it is preferred that themethods of the invention are practised with a single cell type e.g. withmonoclonal cells. Preferably, the cells used in the methods of thepresent invention are from a single cell line. Furthermore, the samecell line may be used for reassorting the virus and for any subsequentpropagation of the virus.

Preferably, the cells are cultured in the absence of serum, to avoid acommon source of contaminants. Various serum-free media for eukaryoticcell culture are known to the person skilled in the art (e.g. Iscove'smedium, ultra CHO medium (BioWhittaker), EX-CELL (JRH Biosciences)).Furthermore, protein-free media may be used (e.g. PF-CHO (JRHBiosciences)). Otherwise, the cells for replication can also be culturedin the customary serum-containing media (e.g. MEM or DMEM medium with0.5% to 10% of fetal calf serum).

The cells may be in adherent culture or in suspension.

Conventional Reassortment

Traditionally, influenza viruses are reassorted by co-infecting aculture host, usually eggs, with a donor strain and a vaccine strain.Reassortant viruses are selected by adding antibodies with specificityfor the HA and/or NA proteins of the donor strain in order to select forreassortant viruses that contain the vaccine strain's HA and/or NAproteins. Over several passages of this treatment one can select forfast growing reassortant viruses containing the vaccine strain's HAand/or NA segments.

The invention is suitable for use in these methods. It can be easier touse vaccine strains with a different HA and/or NA subtype compared tothe donor strain(s) as this facilitates selection for reassortantviruses. It is also possible, however, to use vaccine strains with thesame HA and/or NA subtype as the donor strain(s) and in some aspects ofthe invention this preferred. In this case, antibodies with preferentialspecificity for the HA and/or NA proteins of the donor strain(s) shouldbe available.

Virus Preparation

In one embodiment, the invention provides a method for producinginfluenza viruses comprising steps of (a) infecting a culture host witha reassortant virus of the invention; (b) culturing the host from step(a) to produce the virus; and optionally (c) purifying the virusproduced in step (b).

The culture host may be cells or embryonated hen eggs. Where cells areused as a culture host in this aspect of the invention, it is known thatcell culture conditions (e.g. temperature, cell density, pH value, etc.)are variable over a wide range subject to the cell line and the virusemployed and can be adapted to the requirements of the application. Thefollowing information therefore merely represents guidelines.

As mentioned above, cells are preferably cultured in serum-free orprotein-free media.

Multiplication of the cells can be conducted in accordance with methodsknown to those of skill in the art. For example, the cells can becultivated in a perfusion system using ordinary support methods likecentrifugation or filtration. Moreover, the cells can be multipliedaccording to the invention in a fed-batch system before infection. Inthe context of the present invention, a culture system is referred to asa fed-batch system in which the cells are initially cultured in a batchsystem and depletion of nutrients (or part of the nutrients) in themedium is compensated by controlled feeding of concentrated nutrients.It can be advantageous to adjust the pH value of the medium duringmultiplication of cells before infection to a value between pH 6.6 andpH 7.8 and especially between a value between pH 7.2 and pH 7.3.Culturing of cells preferably occurs at a temperature between 30 and 40°C. When culturing the infected cells (step b), the cells are preferablycultured at a temperature of between 30° C. and 36° C. or between 32° C.and 34° C. or at 33° C. This is particularly preferred, as it has beenshown that incubation of infected cells in this temperature rangeresults in production of a virus that results in improved efficacy whenformulated into a vaccine [27].

Oxygen partial pressure can be adjusted during culturing beforeinfection preferably at a value between 25% and 95% and especially at avalue between 35% and 60%. The values for the oxygen partial pressurestated in the context of the invention are based on saturation of air.Infection of cells occurs at a cell density of preferably about 8-25×10⁵cells/mL in the batch system or preferably about 5-20×10⁶ cells/mL inthe perfusion system. The cells can be infected with a viral dose (MOIvalue, “multiplicity of infection”; corresponds to the number of virusunits per cell at the time of infection) between 10⁻⁸ and 10, preferablybetween 0.0001 and 0.5.

Virus may be grown on cells in adherent culture or in suspension.Microcarrier cultures can be used. In some embodiments, the cells maythus be adapted for growth in suspension.

The methods according to the invention also include harvesting andisolation of viruses or the proteins generated by them. During isolationof viruses or proteins, the cells are separated from the culture mediumby standard methods like separation, filtration or ultrafiltration. Theviruses or the proteins are then concentrated according to methodssufficiently known to those skilled in the art, like gradientcentrifugation, filtration, precipitation, chromatography, etc., andthen purified. It is also preferred according to the invention that theviruses are inactivated during or after purification. Virus inactivationcan occur, for example, by β-propiolactone or formaldehyde at any pointwithin the purification process.

The culture host may be eggs. The current standard method for influenzavirus growth for vaccines uses embryonated SPF hen eggs, with virusbeing purified from the egg contents (allantoic fluid). It is alsopossible to passage a virus through eggs and subsequently propagate itin cell culture and vice versa.

Vaccine

The invention utilises virus produced according to the method to producevaccines.

Vaccines (particularly for influenza virus) are generally based eitheron live virus or on inactivated virus. Inactivated vaccines may be basedon whole virions, ‘split’ virions, or on purified surface antigens.Antigens can also be presented in the form of virosomes. The inventioncan be used for manufacturing any of these types of vaccine.

Where an inactivated virus is used, the vaccine may comprise wholevirion, split virion, or purified surface antigens (for influenza,including hemagglutinin and, usually, also including neuraminidase).Chemical means for inactivating a virus include treatment with aneffective amount of one or more of the following agents: detergents,formaldehyde, β-propiolactone, methylene blue, psoralen,carboxyfullerene (C60), binary ethylamine, acetyl ethyleneimine, orcombinations thereof. Non-chemical methods of viral inactivation areknown in the art, such as for example UV light or gamma irradiation.

Virions can be harvested from virus-containing fluids, e.g. allantoicfluid or cell culture supernatant, by various methods. For example, apurification process may involve zonal centrifugation using a linearsucrose gradient solution that includes detergent to disrupt thevirions. Antigens may then be purified, after optional dilution, bydiafiltration.

Split virions are obtained by treating purified virions with detergents(e.g. ethyl ether, polysorbate 80, deoxycholate, tri-N-butyl phosphate,Triton X-100, Triton N101, cetyltrimethylammonium bromide, Tergitol NP9,etc.) to produce subvirion preparations, including the ‘Tween-ether’splitting process. Methods of splitting influenza viruses, for exampleare well known in the art e.g. see refs. 28-33, etc. Splitting of thevirus is typically carried out by disrupting or fragmenting whole virus,whether infectious or non-infectious with a disrupting concentration ofa splitting agent. The disruption results in a full or partialsolubilisation of the virus proteins, altering the integrity of thevirus. Preferred splitting agents are non-ionic and ionic (e.g.cationic) surfactants e.g. alkylglycosides, alkylthioglycosides, acylsugars, sulphobetaines, betains, polyoxyethylenealkylethers,N,N-dialkyl-Glucamides, Hecameg, alkylphenoxy-polyethoxyethanols, NP9,quaternary ammonium compounds, sarcosyl, CTABs (cetyl trimethyl ammoniumbromides), tri-N-butyl phosphate, Cetavlon, myristyltrimethylammoniumsalts, lipofectin, lipofectamine, and DOT-MA, the octyl- or nonylphenoxypolyoxyethanols (e.g. the Triton surfactants, such as Triton X-100 orTriton N101), polyoxyethylene sorbitan esters (the Tween surfactants),polyoxyethylene ethers, polyoxyethlene esters, etc. One useful splittingprocedure uses the consecutive effects of sodium deoxycholate andformaldehyde, and splitting can take place during initial virionpurification (e.g. in a sucrose density gradient solution). Thus asplitting process can involve clarification of the virion-containingmaterial (to remove non-virion material), concentration of the harvestedvirions (e.g. using an adsorption method, such as CaHPO₄ adsorption),separation of whole virions from non-virion material, splitting ofvirions using a splitting agent in a density gradient centrifugationstep (e.g. using a sucrose gradient that contains a splitting agent suchas sodium deoxycholate), and then filtration (e.g. ultrafiltration) toremove undesired materials. Split virions can usefully be resuspended insodium phosphate-buffered isotonic sodium chloride solution. Examples ofsplit influenza vaccines are the BEGRIVAC™ FLUARIX™, FLUZONE™ andFLUSHIELD™ products.

Purified influenza virus surface antigen vaccines comprise the surfaceantigens hemagglutinin and, typically, also neuraminidase. Processes forpreparing these proteins in purified form are well known in the art. TheFLUVIRIN™, AGRIPPAL™ and INFLUVAC™ products are influenza subunitvaccines.

Another form of inactivated antigen is the virosome [34] (nucleic acidfree viral-like liposomal particles). Virosomes can be prepared bysolubilization of virus with a detergent followed by removal of thenucleocapsid and reconstitution of the membrane containing the viralglycoproteins. An alternative method for preparing virosomes involvesadding viral membrane glycoproteins to excess amounts of phospholipids,to give liposomes with viral proteins in their membrane.

The methods of the invention may also be used to produce live vaccines.Such vaccines are usually prepared by purifying virions fromvirion-containing fluids. For example, the fluids may be clarified bycentrifugation, and stabilized with buffer (e.g. containing sucrose,potassium phosphate, and monosodium glutamate). Various forms ofinfluenza virus vaccine are currently available (e.g. see chapters 17 &18 of reference 35). Live virus vaccines include MedImmune's FLUMIST™product (trivalent live virus vaccine).

The virus may be attenuated. The virus may be temperature-sensitive. Thevirus may be cold-adapted. These three features are particularly usefulwhen using live virus as an antigen.

HA is the main immunogen in current inactivated influenza vaccines, andvaccine doses are standardised by reference to HA levels, typicallymeasured by SRID. Existing vaccines typically contain about 15 μg of HAper strain, although lower doses can be used e.g. for children, or inpandemic situations, or when using an adjuvant. Fractional doses such as½ (i.e. 7.5 μg HA per strain), ¼ and ⅛ have been used, as have higherdoses (e.g. 3× or 9× doses [36,37]). Thus vaccines may include between0.1 and 150 μg of HA per influenza strain, preferably between 0.1 and 50μg e.g. 0.1-20 μg, 0.1-15 μg, 0.1-10 μg, 0.1-7.5 μg, 0.5-5 μg, etc.Particular doses include e.g. about 45, about 30, about 15, about 10,about 7.5, about 5, about 3.8, about 3.75, about 1.9, about 1.5, etc.per strain.

For live vaccines, dosing is measured by median tissue cultureinfectious dose (TCID₅₀) rather than HA content, and a TCID₅₀ of between10⁶ and 10⁸ (preferably between 10^(6.5)-10^(7.5)) per strain istypical.

Influenza strains used with the invention may have a natural HA as foundin a wild-type virus, or a modified HA. For instance, it is known tomodify HA to remove determinants (e.g. hyper-basic regions around theHA1/HA2 cleavage site) that cause a virus to be highly pathogenic inavian species. The use of reverse genetics facilitates suchmodifications.

As well as being suitable for immunizing against inter-pandemic strains,the compositions of the invention are particularly useful for immunizingagainst pandemic or potentially-pandemic strains. The invention issuitable for vaccinating humans as well as non-human animals.

Other strains whose antigens can usefully be included in thecompositions are strains which are resistant to antiviral therapy (e.g.resistant to oseltamivir [38] and/or zanamivir), including resistantpandemic strains [39].

Compositions of the invention may include antigen(s) from one or more(e.g. 1, 2, 3, 4 or more) influenza virus strains, including influenza Avirus and/or influenza B virus provided that at least one influenzastrain is a reassortant influenza strain of the invention. Compositionswherein at least two, at least three or all of the antigens are fromreassortant influenza strains of the invention are also envisioned.Where a vaccine includes more than one strain of influenza, thedifferent strains are typically grown separately and are mixed after theviruses have been harvested and antigens have been prepared. Thus aprocess of the invention may include the step of mixing antigens frommore than one influenza strain. A trivalent vaccine is typical,including antigens from two influenza A virus strains and one influenzaB virus strain. A tetravalent vaccine is also useful [40], includingantigens from two influenza A virus strains and two influenza B virusstrains, or three influenza A virus strains and one influenza B virusstrain.

Pharmaceutical Compositions

Vaccine compositions manufactured according to the invention arepharmaceutically acceptable. They usually include components in additionto the antigens e.g. they typically include one or more pharmaceuticalcarrier(s) and/or excipient(s). As described below, adjuvants may alsobe included. A thorough discussion of such components is available inreference 41.

Vaccine compositions will generally be in aqueous form. However, somevaccines may be in dry form, e.g. in the form of injectable solids ordried or polymerized preparations on a patch.

Vaccine compositions may include preservatives such as thiomersal or2-phenoxyethanol. It is preferred, however, that the vaccine should besubstantially free from (i.e. less than 5 μg/ml) mercurial material e.g.thiomersal-free [32,42]. Vaccines containing no mercury are morepreferred. An α-tocopherol succinate can be included as an alternativeto mercurial compounds [32]. Preservative-free vaccines are particularlypreferred.

To control tonicity, it is preferred to include a physiological salt,such as a sodium salt. Sodium chloride (NaCl) is preferred, which may bepresent at between 1 and 20 mg/ml. Other salts that may be presentinclude potassium chloride, potassium dihydrogen phosphate, disodiumphosphate dehydrate, magnesium chloride, calcium chloride, etc.

Vaccine compositions will generally have an osmolality of between 200mOsm/kg and 400 mOsm/kg, preferably between 240-360 mOsm/kg, and willmore preferably fall within the range of 290-310 mOsm/kg. Osmolality haspreviously been reported not to have an impact on pain caused byvaccination [43], but keeping osmolality in this range is neverthelesspreferred.

Vaccine compositions may include one or more buffers. Typical buffersinclude: a phosphate buffer; a Tris buffer; a borate buffer; a succinatebuffer; a histidine buffer (particularly with an aluminum hydroxideadjuvant); or a citrate buffer. Buffers will typically be included inthe 5-20 mM range.

The pH of a vaccine composition will generally be between 5.0 and 8.1,and more typically between 6.0 and 8.0 e.g. 6.5 and 7.5, or between 7.0and 7.8. A process of the invention may therefore include a step ofadjusting the pH of the bulk vaccine prior to packaging.

The vaccine composition is preferably sterile. The vaccine compositionis preferably non-pyrogenic e.g. containing <1 EU (endotoxin unit, astandard measure) per dose, and preferably <0.1 EU per dose. The vaccinecomposition is preferably gluten-free.

Vaccine compositions of the invention may include detergent e.g. apolyoxyethylene sorbitan ester surfactant (known as ‘Tweens’), anoctoxynol (such as octoxynol-9 (Triton X-100) ort-octylphenoxypolyethoxyethanol), a cetyl trimethyl ammonium bromide(‘CTAB’), or sodium deoxycholate, particularly for a split or surfaceantigen vaccine. The detergent may be present only at trace amounts.Thus the vaccine may include less than 1 mg/ml of each of octoxynol-10and polysorbate 80. Other residual components in trace amounts could beantibiotics (e.g. neomycin, kanamycin, polymyxin B).

A vaccine composition may include material for a single immunisation, ormay include material for multiple immunisations (i.e. a ‘multidose’kit). The inclusion of a preservative is preferred in multidosearrangements. As an alternative (or in addition) to including apreservative in multidose compositions, the compositions may becontained in a container having an aseptic adaptor for removal ofmaterial.

Influenza vaccines are typically administered in a dosage volume ofabout 0.5 ml, although a half dose (i.e. about 0.25 ml) may beadministered to children.

Compositions and kits are preferably stored at between 2° C. and 8° C.They should not be frozen. They should ideally be kept out of directlight.

Host Cell DNA

Where virus has been isolated and/or grown on a cell line, it isstandard practice to minimize the amount of residual cell line DNA inthe final vaccine, in order to minimize any potential oncogenic activityof the DNA.

Thus a vaccine composition prepared according to the inventionpreferably contains less than 10 ng (preferably less than ing, and morepreferably less than 100 pg) of residual host cell DNA per dose,although trace amounts of host cell DNA may be present.

It is preferred that the average length of any residual host cell DNA isless than 500 bp e.g. less than 400 bp, less than 300 bp, less than 200bp, less than 100 bp, etc.

Contaminating DNA can be removed during vaccine preparation usingstandard purification procedures e.g. chromatography, etc. Removal ofresidual host cell DNA can be enhanced by nuclease treatment e.g. byusing a DNase. A convenient method for reducing host cell DNAcontamination is disclosed in references 44 & 45, involving a two-steptreatment, first using a DNase (e.g. Benzonase), which may be usedduring viral growth, and then a cationic detergent (e.g. CTAB), whichmay be used during virion disruption. Treatment with an alkylatingagent, such as β-propiolactone, can also be used to remove host cellDNA, and advantageously may also be used to inactivate virions [46].

Adjuvants

Compositions of the invention may advantageously include an adjuvant,which can function to enhance the immune responses (humoral and/orcellular) elicited in a subject who receives the composition. Preferredadjuvants comprise oil-in-water emulsions. Various such adjuvants areknown, and they typically include at least one oil and at least onesurfactant, with the oil(s) and surfactant(s) being biodegradable(metabolisable) and biocompatible. The oil droplets in the emulsion aregenerally less than 5 μm in diameter, and ideally have a sub-microndiameter, with these small sizes being achieved with a microfluidiser toprovide stable emulsions. Droplets with a size less than 220 nm arepreferred as they can be subjected to filter sterilization.

The emulsion can comprise oils such as those from an animal (such asfish) or vegetable source. Sources for vegetable oils include nuts,seeds and grains. Peanut oil, soybean oil, coconut oil, and olive oil,the most commonly available, exemplify the nut oils. Jojoba oil can beused e.g. obtained from the jojoba bean. Seed oils include saffloweroil, cottonseed oil, sunflower seed oil, sesame seed oil and the like.In the grain group, corn oil is the most readily available, but the oilof other cereal grains such as wheat, oats, rye, rice, teff, triticaleand the like may also be used. 6-10 carbon fatty acid esters of glyceroland 1,2-propanediol, while not occurring naturally in seed oils, may beprepared by hydrolysis, separation and esterification of the appropriatematerials starting from the nut and seed oils. Fats and oils frommammalian milk are metabolizable and may therefore be used in thepractice of this invention. The procedures for separation, purification,saponification and other means necessary for obtaining pure oils fromanimal sources are well known in the art. Most fish containmetabolizable oils which may be readily recovered. For example, codliver oil, shark liver oils, and whale oil such as spermaceti exemplifyseveral of the fish oils which may be used herein. A number of branchedchain oils are synthesized biochemically in 5-carbon isoprene units andare generally referred to as terpenoids. Shark liver oil contains abranched, unsaturated terpenoids known as squalene,2,6,10,15,19,23-hexamethyl-2,6,10,14,18,22-tetracosahexaene, which isparticularly preferred herein. Squalane, the saturated analog tosqualene, is also a preferred oil. Fish oils, including squalene andsqualane, are readily available from commercial sources or may beobtained by methods known in the art. Another preferred oil isα-tocopherol (see below).

Mixtures of oils can be used.

Surfactants can be classified by their ‘HLB’ (hydrophile/lipophilebalance). Preferred surfactants of the invention have a HLB of at least10, preferably at least 15, and more preferably at least 16. Theinvention can be used with surfactants including, but not limited to:the polyoxyethylene sorbitan esters surfactants (commonly referred to asthe Tweens), especially polysorbate 20 and polysorbate 80; copolymers ofethylene oxide (EO), propylene oxide (PO), and/or butylene oxide (BO),sold under the DOWFAX™ tradename, such as linear EO/PO block copolymers;octoxynols, which can vary in the number of repeating ethoxy(oxy-1,2-ethanediyl) groups, with octoxynol-9 (Triton X-100, ort-octylphenoxypolyethoxyethanol) being of particular interest;(octylphenoxy)polyethoxyethanol (IGEPAL CA-630/NP-40); phospholipidssuch as phosphatidylcholine (lecithin); nonylphenol ethoxylates, such asthe Tergitol™ NP series; polyoxyethylene fatty ethers derived fromlauryl, cetyl, stearyl and oleyl alcohols (known as Brij surfactants),such as triethyleneglycol monolauryl ether (Brij 30); and sorbitanesters (commonly known as the SPANs), such as sorbitan trioleate (Span85) and sorbitan monolaurate. Non-ionic surfactants are preferred.Preferred surfactants for including in the emulsion are Tween 80(polyoxyethylene sorbitan monooleate), Span 85 (sorbitan trioleate),lecithin and Triton X-100.

Mixtures of surfactants can be used e.g. Tween 80/Span 85 mixtures. Acombination of a polyoxyethylene sorbitan ester such as polyoxyethylenesorbitan monooleate (Tween 80) and an octoxynol such ast-octylphenoxypolyethoxyethanol (Triton X-100) is also suitable. Anotheruseful combination comprises laureth 9 plus a polyoxyethylene sorbitanester and/or an octoxynol.

Preferred amounts of surfactants (% by weight) are: polyoxyethylenesorbitan esters (such as Tween 80) 0.01 to 1%, in particular about 0.1%;octyl- or nonylphenoxy polyoxyethanols (such as Triton X-100, or otherdetergents in the Triton series) 0.001 to 0.1%, in particular 0.005 to0.02%; polyoxyethylene ethers (such as laureth 9) 0.1 to 20%, preferably0.1 to 10% and in particular 0.1 to 1% or about 0.5%.

Where the vaccine contains a split virus, it is preferred that itcontains free surfactant in the aqueous phase. This is advantageous asthe free surfactant can exert a ‘splitting effect’ on the antigen,thereby disrupting any unsplit virions and/or virion aggregates thatmight otherwise be present. This can improve the safety of split virusvaccines [47].

Preferred emulsions have an average droplets size of <1 μm e.g. ≦750 nm,≦500 nm, ≦400 nm, ≦300 nm, ≦250 nm, ≦220 nm, ≦200 nm, or smaller. Thesedroplet sizes can conveniently be achieved by techniques such asmicrofluidisation.

Specific oil-in-water emulsion adjuvants useful with the inventioninclude, but are not limited to:

-   -   A submicron emulsion of squalene, Tween 80, and Span 85. The        composition of the emulsion by volume can be about 5% squalene,        about 0.5% polysorbate 80 and about 0.5% Span 85. In weight        terms, these ratios become 4.3% squalene, 0.5% polysorbate 80        and 0.48% Span 85. This adjuvant is known as ‘MF59’ [48-50], as        described in more detail in Chapter 10 of ref. 51 and chapter 12        of ref. 52. The MF59 emulsion advantageously includes citrate        ions e.g. 10 mM sodium citrate buffer.    -   An emulsion comprising squalene, a tocopherol, and        polysorbate 80. The emulsion may include phosphate buffered        saline. These emulsions may have by volume from 2 to 10%        squalene, from 2 to 10% tocopherol and from 0.3 to 3%        polysorbate 80, and the weight ratio of squalene:tocopherol is        preferably <1 (e.g. 0.90) as this can provide a more stable        emulsion. Squalene and polysorbate 80 may be present in a volume        ratio of about 5:2 or at a weight ratio of about 11:5. Thus the        three components (squalene, tocopherol, polysorbate 80) may be        present at a weight ratio of 1068:1186:485 or around 55:61:25.        One such emulsion (‘AS03’) can be made by dissolving Tween 80 in        PBS to give a 2% solution, then mixing 90 ml of this solution        with a mixture of (5 g of DL a tocopherol and 5 ml squalene),        then microfluidising the mixture. The resulting emulsion may        have submicron oil droplets e.g. with an average diameter of        between 100 and 250 nm, preferably about 180 nm. The emulsion        may also include a 3-de-O-acylated monophosphoryl lipid A (3d        MPL). Another useful emulsion of this type may comprise, per        human dose, 0.5-10 mg squalene, 0.5-11 mg tocopherol, and 0.1-4        mg polysorbate 80 [53] e.g. in the ratios discussed above.    -   An emulsion of squalene, a tocopherol, and a Triton detergent        (e.g. Triton X-100). The emulsion may also include a 3d-MPL (see        below). The emulsion may contain a phosphate buffer.    -   An emulsion comprising a polysorbate (e.g. polysorbate 80), a        Triton detergent (e.g. Triton X-100) and a tocopherol (e.g. an        α-tocopherol succinate). The emulsion may include these three        components at a mass ratio of about 75:11:10 (e.g. 750 μg/ml        polysorbate 80, 110 μg/ml Triton X-100 and 100 μg/ml        α-tocopherol succinate), and these concentrations should include        any contribution of these components from antigens. The emulsion        may also include squalene. The emulsion may also include a        3d-MPL (see below). The aqueous phase may contain a phosphate        buffer.    -   An emulsion of squalane, polysorbate 80 and poloxamer 401        (“Pluronic™ L121”). The emulsion can be formulated in phosphate        buffered saline, pH 7.4. This emulsion is a useful delivery        vehicle for muramyl dipeptides, and has been used with        threonyl-MDP in the “SAF-1” adjuvant [54] (0.05-1% Thr-MDP, 5%        squalane, 2.5% Pluronic L121 and 0.2% polysorbate 80). It can        also be used without the Thr-MDP, as in the “AF” adjuvant [55]        (5% squalane, 1.25% Pluronic L121 and 0.2% polysorbate 80).        Microfluidisation is preferred.    -   An emulsion comprising squalene, an aqueous solvent, a        polyoxyethylene alkyl ether hydrophilic nonionic surfactant        (e.g. polyoxyethylene (12) cetostearyl ether) and a hydrophobic        nonionic surfactant (e.g. a sorbitan ester or mannide ester,        such as sorbitan monoleate or ‘Span 80’). The emulsion is        preferably thermoreversible and/or has at least 90% of the oil        droplets (by volume) with a size less than 200 nm [56]. The        emulsion may also include one or more of: alditol; a        cryoprotective agent (e.g. a sugar, such as dodecylmaltoside        and/or sucrose); and/or an alkylpolyglycoside. The emulsion may        include a TLR4 agonist [57]. Such emulsions may be lyophilized.    -   An emulsion of squalene, poloxamer 105 and Abil-Care [58]. The        final concentration (weight) of these components in adjuvanted        vaccines are 5% squalene, 4% poloxamer 105 (pluronic polyol) and        2% Abil-Care 85 (Bis-PEG/PPG-16/16 PEG/PPG-16/16 dimethicone;        caprylic/capric triglyceride).    -   An emulsion having from 0.5-50% of an oil, 0.1-10% of a        phospholipid, and 0.05-5% of a non-ionic surfactant. As        described in reference 59, preferred phospholipid components are        phosphatidylcholine, phosphatidylethanolamine,        phosphatidylserine, phosphatidylinositol, phosphatidylglycerol,        phosphatidic acid, sphingomyelin and cardiolipin. Submicron        droplet sizes are advantageous.    -   A submicron oil-in-water emulsion of a non-metabolisable oil        (such as light mineral oil) and at least one surfactant (such as        lecithin, Tween 80 or Span 80). Additives may be included, such        as QuilA saponin, cholesterol, a saponin-lipophile conjugate        (such as GPI-0100, described in reference 60, produced by        addition of aliphatic amine to desacylsaponin via the carboxyl        group of glucuronic acid), dimethyidioctadecylammonium bromide        and/or N,N-dioctadecyl-N,N-bis (2-hydroxyethyl)propanediamine.    -   An emulsion in which a saponin (e.g. QuilA or QS21) and a sterol        (e.g. a cholesterol) are associated as helical micelles [61].    -   An emulsion comprising a mineral oil, a non-ionic lipophilic        ethoxylated fatty alcohol, and a non-ionic hydrophilic        surfactant (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [62].    -   An emulsion comprising a mineral oil, a non-ionic hydrophilic        ethoxylated fatty alcohol, and a non-ionic lipophilic surfactant        (e.g. an ethoxylated fatty alcohol and/or        polyoxyethylene-polyoxypropylene block copolymer) [62].

In some embodiments an emulsion may be mixed with antigenextemporaneously, at the time of delivery, and thus the adjuvant andantigen may be kept separately in a packaged or distributed vaccine,ready for final formulation at the time of use. In other embodiments anemulsion is mixed with antigen during manufacture, and thus thecomposition is packaged in a liquid adjuvanted form. The antigen willgenerally be in an aqueous form, such that the vaccine is finallyprepared by mixing two liquids. The volume ratio of the two liquids formixing can vary (e.g. between 5:1 and 1:5) but is generally about 1:1.Where concentrations of components are given in the above descriptionsof specific emulsions, these concentrations are typically for anundiluted composition, and the concentration after mixing with anantigen solution will thus decrease.

Packaging of Vaccine Compositions

Suitable containers for compositions of the invention (or kitcomponents) include vials, syringes (e.g. disposable syringes), nasalsprays, etc. These containers should be sterile.

Where a composition/component is located in a vial, the vial ispreferably made of a glass or plastic material. The vial is preferablysterilized before the composition is added to it. To avoid problems withlatex-sensitive patients, vials are preferably sealed with a latex-freestopper, and the absence of latex in all packaging material ispreferred. The vial may include a single dose of vaccine, or it mayinclude more than one dose (a ‘multidose’ vial) e.g. 10 doses. Preferredvials are made of colourless glass.

A vial can have a cap (e.g. a Luer lock) adapted such that a pre-filledsyringe can be inserted into the cap, the contents of the syringe can beexpelled into the vial (e.g. to reconstitute lyophilised materialtherein), and the contents of the vial can be removed back into thesyringe. After removal of the syringe from the vial, a needle can thenbe attached and the composition can be administered to a patient. Thecap is preferably located inside a seal or cover, such that the seal orcover has to be removed before the cap can be accessed. A vial may havea cap that permits aseptic removal of its contents, particularly formultidose vials.

Where a component is packaged into a syringe, the syringe may have aneedle attached to it. If a needle is not attached, a separate needlemay be supplied with the syringe for assembly and use. Such a needle maybe sheathed. Safety needles are preferred. 1-inch 23-gauge, 1-inch25-gauge and 5/8-inch 25-gauge needles are typical. Syringes may beprovided with peel-off labels on which the lot number, influenza seasonand expiration date of the contents may be printed, to facilitate recordkeeping. The plunger in the syringe preferably has a stopper to preventthe plunger from being accidentally removed during aspiration. Thesyringes may have a latex rubber cap and/or plunger. Disposable syringescontain a single dose of vaccine. The syringe will generally have a tipcap to seal the tip prior to attachment of a needle, and the tip cap ispreferably made of a butyl rubber. If the syringe and needle arepackaged separately then the needle is preferably fitted with a butylrubber shield. Preferred syringes are those marketed under the tradename “Tip-Lok”™.

Containers may be marked to show a half-dose volume e.g. to facilitatedelivery to children. For instance, a syringe containing a 0.5 ml dosemay have a mark showing a 0.25 ml volume.

Where a glass container (e.g. a syringe or a vial) is used, then it ispreferred to use a container made from a borosilicate glass rather thanfrom a soda lime glass.

A kit or composition may be packaged (e.g. in the same box) with aleaflet including details of the vaccine e.g. instructions foradministration, details of the antigens within the vaccine, etc. Theinstructions may also contain warnings e.g. to keep a solution ofadrenaline readily available in case of anaphylactic reaction followingvaccination, etc.

Methods of Treatment, and Administration of the Vaccine

The invention provides a vaccine manufactured according to theinvention. These vaccine compositions are suitable for administration tohuman or non-human animal subjects, such as pigs or birds, and theinvention provides a method of raising an immune response in a subject,comprising the step of administering a composition of the invention tothe subject. The invention also provides a composition of the inventionfor use as a medicament, and provides the use of a composition of theinvention for the manufacture of a medicament for raising an immuneresponse in a subject.

The immune response raised by these methods and uses will generallyinclude an antibody response, preferably a protective antibody response.Methods for assessing antibody responses, neutralising capability andprotection after influenza virus vaccination are well known in the art.Human studies have shown that antibody titers against hemagglutinin ofhuman influenza virus are correlated with protection (a serum samplehemagglutination-inhibition titer of about 30-40 gives around 50%protection from infection by a homologous virus) [63]. Antibodyresponses are typically measured by hemagglutination inhibition, bymicroneutralisation, by single radial immunodiffusion (SRID), and/or bysingle radial hemolysis (SRH). These assay techniques are well known inthe art.

Compositions of the invention can be administered in various ways. Themost preferred immunisation route is by intramuscular injection (e.g.into the arm or leg), but other available routes include subcutaneousinjection, intranasal [64-66], oral [67], intradermal [68,69],transcutaneous, transdermal [70], etc.

Vaccines prepared according to the invention may be used to treat bothchildren and adults. Influenza vaccines are currently recommended foruse in pediatric and adult immunisation, from the age of 6 months. Thusa human subject may be less than 1 year old, 1-5 years old, 5-15 yearsold, 15-55 years old, or at least 55 years old. Preferred subjects forreceiving the vaccines are the elderly (e.g. ≧50 years old, ≧60 yearsold, and preferably ≧65 years), the young (e.g. <5 years old),hospitalised subjects, healthcare workers, armed service and militarypersonnel, pregnant women, the chronically ill, immunodeficientsubjects, subjects who have taken an antiviral compound (e.g. anoseltamivir or zanamivir compound; see below) in the 7 days prior toreceiving the vaccine, people with egg allergies and people travellingabroad. The vaccines are not suitable solely for these groups, however,and may be used more generally in a population. For pandemic strains,administration to all age groups is preferred.

Preferred compositions of the invention satisfy 1, 2 or 3 of the CPMPcriteria for efficacy. In adults (18-60 years), these criteria are: (1)≧70% seroprotection; (2) ≧40% seroconversion; and/or (3) a GMT increaseof ≧2.5-fold. In elderly (≧60 years), these criteria are: (1) ≧60%seroprotection; (2) ≧30% seroconversion; and/or (3) a GMT increase of≧2-fold. These criteria are based on open label studies with at least 50patients.

Treatment can be by a single dose schedule or a multiple dose schedule.Multiple doses may be used in a primary immunisation schedule and/or ina booster immunisation schedule. In a multiple dose schedule the variousdoses may be given by the same or different routes e.g. a parenteralprime and mucosal boost, a mucosal prime and parenteral boost, etc.Administration of more than one dose (typically two doses) isparticularly useful in immunologically naïve patients e.g. for peoplewho have never received an influenza vaccine before, or for vaccinatingagainst a new HA subtype (as in a pandemic outbreak). Multiple doseswill typically be administered at least 1 week apart (e.g. about 2weeks, about 3 weeks, about 4 weeks, about 6 weeks, about 8 weeks, about10 weeks, about 12 weeks, about 16 weeks, etc.).

Vaccines produced by the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional or vaccinationcentre) other vaccines e.g. at substantially the same time as a measlesvaccine, a mumps vaccine, a rubella vaccine, a MMR vaccine, a varicellavaccine, a MMRV vaccine, a diphtheria vaccine, a tetanus vaccine, apertussis vaccine, a DTP vaccine, a conjugated H. influenzae type bvaccine, an inactivated poliovirus vaccine, a hepatitis B virus vaccine,a meningococcal conjugate vaccine (such as a tetravalent A-C-W135-Yvaccine), a respiratory syncytial virus vaccine, a pneumococcalconjugate vaccine, etc. Administration at substantially the same time asa pneumococcal vaccine and/or a meningococcal vaccine is particularlyuseful in elderly patients.

Similarly, vaccines of the invention may be administered to patients atsubstantially the same time as (e.g. during the same medicalconsultation or visit to a healthcare professional) an antiviralcompound, and in particular an antiviral compound active againstinfluenza virus (e.g. oseltamivir and/or zanamivir). These antiviralsinclude neuraminidase inhibitors, such as a(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid or5-(acetylamino)-4-[(aminoiminomethyl)-amino]-2,6-anhydro-3,4,5-trideoxy-D-glycero-D-galactonon-2-enonicacid, including esters thereof (e.g. the ethyl esters) and salts thereof(e.g. the phosphate salts). A preferred antiviral is(3R,4R,5S)-4-acetylamino-5-amino-3(1-ethylpropoxy)-1-cyclohexene-1-carboxylicacid, ethyl ester, phosphate (1:1), also known as oseltamivir phosphate(TAMIFLU™).

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The term “about” in relation to a numerical value x is optional andmeans, for example, x±10%.

Unless specifically stated, a process comprising a step of mixing two ormore components does not require any specific order of mixing. Thuscomponents can be mixed in any order. Where there are three componentsthen two components can be combined with each other, and then thecombination may be combined with the third component, etc.

The various steps of the methods may be carried out at the same ordifferent times, in the same or different geographical locations, e.g.countries, and by the same or different people or entities.

Where animal (and particularly bovine) materials are used in the cultureof cells, they should be obtained from sources that are free fromtransmissible spongiform encephalopathies (TSEs), and in particular freefrom bovine spongiform encephalopathy (BSE). Overall, it is preferred toculture cells in the total absence of animal-derived materials.

Where a compound is administered to the body as part of a compositionthen that compound may alternatively be replaced by a suitable prodrug.

References to a percentage sequence identity between two amino acidsequences means that, when aligned, that percentage of amino acids arethe same in comparing the two sequences. This alignment and the percenthomology or sequence identity can be determined using software programsknown in the art, for example those described in section 7.7.18 ofreference 71. A preferred alignment is determined by the Smith-Watermanhomology search algorithm using an affine gap search with a gap openpenalty of 12 and a gap extension penalty of 2, BLOSUM matrix of 62. TheSmith-Waterman homology search algorithm is taught in reference 72.

References to a percentage sequence identity between two nucleic acidsequences mean that, when aligned, that percentage of bases are the samein comparing the two sequences. This alignment and the percent homologyor sequence identity can be determined using software programs known inthe art, for example those described in section 7.7.18 of reference 71.A preferred alignment program is GCG Gap (Genetics Computer Group,Wisconsin, Suite Version 10.1), preferably using default parameters,which are as follows: open gap=3; extend gap=1.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 compares the HA content (determined by lectin-capture ELISA) ofsucrose gradient-purified viruses harvested at 60 h post-infection fromMDCK cell cultures infected with reverse genetics-derived 6:2reassortants containing either the PR8-X or #21 backbone with the HA andNA segments from (A) a pandemic-like H1 strain (strain 1) or (B) asecond pandemic-like strain (strain 2). In FIGS. 1A and 1B, the whitebar represents a reference vaccine strain (derived fromWHO-Collaborating Centre-supplied strain) as control, the dotted barrepresents a reassortant virus containing the PR8-X backbone, and thechecked bar represents a reassortant virus containing the #21 backbone.The y-axis indicates HA yield in μg/ml.

FIG. 2 compares the HA content (determined by a lectin-capture ELISA) ofunpurified viruses harvested at 60 h post-infection from MDCK cellcultures infected with reverse genetics-derived 6:2 reassortantscontaining either the PR8-X or #21 backbone with the HA and NA segmentsfrom (A) a pre-pandemic H1 strain (strain 1) and (B) a secondpre-pandemic H1 strain (strain 2). In FIGS. 2A and 2B, the white barrepresents a reference vaccine strain (derived from WHO-CollaboratingCentre-supplied strain) as control, the dotted bar represents areassortant virus containing the PR8-X backbone, and the checked barrepresents a reassortant virus containing the #21 backbone. The y-axisindicates HA yield in μg/ml.

FIG. 3 compares the HA yield (determined by HPLC) of sucrose-purifiedviruses harvested at 60 h post-infection from MDCK cell culturesinfected with reverse genetics-derived 6:2 reassortants containingeither the PR8-X or #21 backbone with the HA and NA segments from an H3strain (strain 1). The white bar represents a reference vaccine strain(derived from WHO-Collaborating Centre-supplied strain) as control, thedotted bar represents a reassortant virus containing the PR8-X backbone,and the checked bar represents a reassortant virus containing the #21backbone. The y-axis indicates HA yield in μg/ml.

FIG. 4 compares virus titers (determined by focus formation assay (FFA);FIG. 4A) and HA titers (determined by lectin-capture ELISA; FIG. 4B) ofviruses harvested from embryonated chicken eggs at 60 h post-infectionwith a reference vaccine strain or reverse genetics-derived 6:2reassortant viruses made with either the PR8-X or #21 backbone and theHA and NA segments from a pandemic-like H1 strain (strain 2). In FIG.4A, the individual dots represent data from single eggs. The linerepresents the average of the individual data points. The y-axisindicates infectious units/ml. In FIG. 4B, the white bar represents thereference vaccine strain (derived from WHO-Collaborating Centre-suppliedstrain), the dotted bar represents a reassortant virus containing thePR8-X backbone, and the checked bar represents a reassortant viruscontaining the #21 backbone. The y-axis indicates HA yield in μg/ml forpooled egg samples

FIG. 5 compares virus titers (determined by FFA; FIG. 5A) and HA titers(determined by lectin-capture ELISA; FIG. 5B) from viruses harvested at60 h post-infection from MDCK cells infected with a reference vaccinestrain or reverse genetics-derived 6:2 reassortant viruses made witheither the #21 or #21C backbone and the HA and NA segments from apandemic-like H1 strain (strain 2). In both figures, the white barrepresents a reference vaccine strain (derived from WHO-CollaboratingCentre-supplied strain) as control, the dotted bar represents areassortant virus made with the #21 backbone, and the checked barrepresents a reassortant virus made with a modified #21 backbone (#21C)containing two canine-adapted mutations (R389K, T559N) in the PR8-X PB2segment that comprises the backbone. The y-axis in FIGS. 5A and 5Bindicates infectious units/ml and HA yield in μg/ml, respectively.

FIG. 6 compares virus titers (determined by FFA) from viruses harvestedat 60 h post-infection from MDCK cells infected with reversegenetics-derived 6:2 reassortant viruses made with either the PR8-X, #21or #21C backbone and the HA and NA segments from a differentpandemic-like H1 strain (strain 1). The white bar represents the PR8-Xbackbone, the dotted bar represents the #21 backbone, and the checkedbar represents the #21C backbone containing two canine-adapted mutations(R389K, T559N) in the PR8-X PB2 segment that comprises the backbone. They-axis indicates infectious units/ml.

FIG. 7 compares HA titers (determined by red blood cell hemagglutinationassay) from viruses harvested at 60 h post-infection from embryonatedchicken eggs infected with a reference vaccine strain (derived fromWHO-Collaborating Centre-supplied strain) or reverse genetics-derived6:2 reassortant viruses containing either the PR8-X or #21C backbone andthe HA and NA segments from a pandemic-like H1 strain (strain 1). Theindividual dots represent data from a single egg. The line representsthe average of the individual data points. The y-axis indicates HAunits.

FIG. 8 compares infectious titers (determined by FFA) of virusesharvested at different time points post-infection of MDCK cells infectedwith reverse genetics-derived 6:2 reassortants made with either a PR8-Xbackbone or a modified PR8-X backbone containing canine-adaptedpolymerase mutations and the HA and NA segments from a pandemic-like H1strain (strain 1). In FIG. 8A, the dotted line with triangle markersindicates the PR8-X backbone and the solid line with square markersindicates a modified PR8-X backbone “PR8-X(cPA)” containing threecanine-adapted mutations (E327K, N444D, and N675D) in the PR8-X PAsegment. In FIG. 8B, the dotted line with triangle markers indicates thePR8-X backbone and the solid line with open circle markers indicates amodified PR8-X backbone “PR8-X(cNP)” containing two canine-adaptedmutations (A27T, E375N) in the PR8-X NP segment. In both figures, thex-axis indicates hours post-infection and the y-axis indicatesinfectious units/ml.

FIG. 9 compares infectious titers (determined by FFA; FIG. 9A) and HAtiters (determined by red blood cell hemagglutination assay; FIG. 9B) ofvirus harvested at different times post-infection from MDCK cellsinfected with a reference vaccine strain or reverse genetics-derived 6:2reassortant viruses made with either the PR8-X or modified PR8-Xbackbones containing canine-adapted mutations and the HA and NA segmentsfrom an H3 strain (strain 2). In FIG. 9A, the dotted line with x markersindicates the reference vaccine strain (derived from WHO-CollaboratingCentre-supplied strain), the dotted line with triangle markers indicatesthe PR8-X backbone, the solid line with square markers indicates amodified PR8-X backbone “PR8-X (cPA)” containing three canine-adaptedmutations (E327K, N444D, and N675D) in the PR8-X PA segment, and thesolid line with open circle markers indicates a modified PR8-X backbone“PR8-X (cNP)” containing two canine-adapted mutations a in the PR8-X NPsegment. The y-axis represents infectious units/ml and the x-axisrepresents hours post-infection. In FIG. 9B, the white bar indicates thereference vaccine strain (derived from WHO-Collaborating Centre-suppliedstrain), the dotted bar indicates the PR8-X backbone, the checked barindicates the PR8-X(cPA) backbone and the cross-hatched bar indicatesthe PR8-X(cNP) backbone. The y-axis represents HA units from the 60 hpost-infection time-point.

FIG. 10 compares the HA content (determined by lectin-capture ELISA) ofsucrose gradient-purified viruses harvested at 60 h post-infection fromMDCK cell cultures infected with reverse genetics-derived 6:2reassortants containing either the PR8-X or #21 backbone with the HA andNA segments from (A) an H3 (strain 2) or (B) a second H3 strain (strain3) or (C) a third H3 strain (strain 4). In FIGS. 10A and 10B, the whitebar represents a reference vaccine strain (derived fromWHO-Collaborating Centre-supplied strain) as control, the dotted barrepresents a reassortant virus containing the PR8-X backbone, and thechecked bar represents a reassortant virus containing the #21 backbone.The y-axis indicates HA yield in μg/ml.

MODES FOR CARRYING OUT THE INVENTION

Development of New Donor Strains

In order to provide high-growth donor strains, the inventors found thata reassortant influenza virus comprising the PB1 segment ofA/California/07/09 and all other backbone segments from PR8-X showsimproved growth characteristics compared with reassortant influenzaviruses which contain all backbone segments from PR8-X. This influenzabackbone is referred to as #21.

Focus-Forming Assays (FFA)

For the FFA, uninfected MDCK cells are plated at a density of 1.8×10⁴cells/well in 96 well plates in 100 μl of DMEM with 10% FCS. The nextday, medium is aspirated and cells are infected with viruses in a volumeof 50 μl (viruses diluted in DMEM+1% FCS). The cells are incubated at37° C. until the next day.

At several time points after infection, the medium is aspirated and thecells washed once with PBS. 50 μl of ice-cold 50%/50% acetone-methanolis added to each well followed by incubation at −20° C. for 30 minutes.The acetone mix is aspirated and the cells washed once with PBST(PBS+0.1% Tween). 50 μl of 2% BSA in PBS is added to each well followedby incubation at room temperature (RT) for 30 minutes. 50 μl of a 1:6000dilution of anti-NP is added in blocking buffer followed by incubationat RT for 1 hours. The antibody solution is aspirated and the cellswashed three times with PBST. Secondary antibody (goat anti mouse) isadded at a dilution 1:2000 in 50 μl blocking buffer and the plate isincubated at RT for 1 hours. The antibody solution is aspirated and thecells washed three times with PBST. 50 μl of KPL True Blue is added toeach well and incubated for 10 minutes. The reaction is stopped byaspirating the True-Blue and washing once with dH₂O. The water isaspirated and the cells are left to dry.

Growth Characteristics of Reassortant Viruses Containing PR8-X or #21Backbones

In order to test the suitability of the #21 strain as a donor strain forvirus reassortment, reassortant influenza viruses are produced byreverse genetics which contain the HA and NA proteins from variousinfluenza strains (including zoonotic, seasonal, and pandemic-likestrains) and the other viral segments from either PR8-X or the #21backbone. The HA content, HA yield and the viral titres of thesereassortant viruses are determined. As a control a reference vaccinestrain which does not contain any backbone segments from PR8-X orA/California/07109 is used. These viruses are cultured either inembryonated chicken eggs or in MDCK cells.

The results indicate that reassortant viruses which contain the #21backbone consistently give higher viral titres and HA yields comparedwith the control virus and the virus which contains all backbonesegments from PR8-X in both eggs and cell culture. This difference isdue to the PB1 segment because this is the only difference between #21reassortants and PR8-X reassortants (see FIGS. 1 to 4).

Growth Characteristics of Reassortant Viruses Containing PR8-X or CanineAdapted PR8-X Backbones

In order to test the effect of canine-adapted mutations on the growthcharacteristics of PR8-X, the inventors introduce mutations into the PAsegment (E327K, N444D, and N675D), or the NP segment (A27T, E375N) ofPR8-X. These backbones are referred to as PR8-X(cPA) and PR8-X(cNP),respectively. Reassortant influenza viruses are produced containing thePR8-X(cPA) and PR8-X(cNP) backbones and the HA and NA segments of apandemic-like H1 influenza strain (strain 1) or a H3 influenza strain(strain 2). As a control a reference vaccine strain which does notcontain any backbone segments from PR8-X is used. The reassortantinfluenza viruses are cultured in MDCK cells.

The results show that reassortant influenza viruses which containcanine-adapted backbone segments consistently grow to higher viraltitres compared with reassortant influenza viruses which containunmodified PR8-X backbone segments (see FIGS. 8 and 9).

Growth Characteristics of Reassortant Viruses Containing PR8-X, #21 or#21C Backbones

In order to test whether canine-adapted mutations in the backbonesegments improve the growth characteristics of the #21 backbone, theinventors modify the #21 backbone by introducing mutations into thePR8-X PB2 segment (R389K, T559N). This backbone is referred to as #2 IC.Reassortant influenza viruses are produced by reverse genetics whichcontain the HA and NA proteins from two different pandemic-like H1strains (strains 1 and 2) and the other viral segments from eitherPR8-X, the #21 backbone or the #21C backbone. As a control a referencevaccine strain which does not contain any backbone segments from PR8-Xor A/California/07/09 is used. These viruses are cultured in MDCK cells.The virus yield of these reassortant viruses is determined. Forreassortant influenza viruses containing the HA and NA segments from thepandemic-like H1 strain (strain 1) and the PR8-X or #21C backbones theHA titres are also determined.

The results show that reassortant influenza viruses which contain the#21C backbone consistently grow to higher viral titres compared withreassortant influenza viruses which contain only PR8-X backbone segmentsor the #21 backbone (see FIGS. 5, 6 and 7). Reassortant influenzaviruses comprising the #21C backbone also show higher HA titres comparedwith PR8-X reassortants.

It will be understood that the invention has been described by way ofexample only and modifications may be made whilst remaining within thescope and spirit of the invention.

REFERENCES

-   [1] WO2007/002008-   [2] WO2007/124327-   [3] WO2010/070098-   [4] Needleman & Wunsch (1970) J. Mol. Biol. 48, 443-453.-   [5] Rice et al. (2000) Trends Genet 16:276-277.-   [6] U.S. Pat. No. 6,468,544.-   [7] Herlocher et al. (2004) J Infect Dis 190(9):1627-30.-   [8] Le et al. (2005) Nature 437(7062):1108.-   [9] Neumann et al. (2005) Proc Natl Acad Sci USA 102: 16825-9-   [10] WO2010/133964-   [11] WO2009/000891-   [12] U.S. provisional application No. 61/273,151-   [13] Sambrook et al, Molecular Cloning: A Laboratory Manual, 2 ed.,    1989, Cold Spring Harbor Press, Cold Spring Harbor, N. Y-   [14] WO2011/012999-   [15] Kistner et al. (1998) Vaccine 16:960-8.-   [16] Kistner et al. (1999) Dev Biol Stand 98:101-110.-   [17] Bruhl et al. (2000) Vaccine 19:1149-58.-   [18] Pau et al. (2001) Vaccine 19:2716-21.-   [19] http://www.atcc.org/-   [20] http://locus.umdnj.edu/-   [21] WO97/37000.-   [22] Brands et al. (1999) Dev Biol Stand 98:93-100.-   [23] Halperin et al. (2002) Vaccine 20:1240-7.-   [24] EP-A-1260581 (WO01/64846)-   [25] WO2006/071563-   [26] WO2005/113758-   [27] WO97/37001-   [28] WO02/28422.-   [29] WO02/067983.-   [30] WO02/074336.-   [31] WO01/21151.-   [32] WO02/097072.-   [33] WO2005/113756.-   [34] Huckriede et al. (2003) Methods Enzymol 373:74-91.-   [35] Vaccines. (eds. Plotkins & Orenstein). 4th edition, 2004, ISBN:    0-7216-9688-0-   [36] Treanor et al. (1996) J Infect Dis 173:1467-70.-   [37] Keitel et al. (1996) Clin Diagn Lab Immunol 3:507-10.-   [38] Herlocher et al. (2004) J Infect Dis 190(9):1627-30.-   [39] Le et al. (2005) Nature 437(7062):1108.-   [40] WO2008/068631.-   [41] Gennaro (2000) Remington: The Science and Practice of Pharmacy.    20th edition, ISBN: 0683306472.-   [42] Banzhoff (2000) Immunology Letters 71:91-96.-   [43] Nony et al. (2001) Vaccine 27:3645-51.-   [44] EP-B-0870508.-   [45] U.S. Pat. No. 5,948,410.-   [46] WO2007/052163.-   [47] WO2007/052061-   [48] WO90/14837.-   [49] Podda & Del Giudice (2003) Expert Rev Vaccines 2:197-203.-   [50] Podda (2001) Vaccine 19: 2673-2680.-   [51] Vaccine Design: The Subunit and Adjuvant Approach (eds. Powell    & Newman) Plenum Press 1995 (ISBN 0-306-44867-X).-   [52] Vaccine Adjuvants: Preparation Methods and Research Protocols    (Volume 42 of Methods in Molecular Medicine series). ISBN:    1-59259-083-7. Ed. O'Hagan.-   [53] WO2008/043774.-   [54] Allison & Byars (1992) Res Immunol 143:519-25.-   [55] Hariharan et al. (1995) Cancer Res 55:3486-9.-   [56] US-2007/014805.-   [57] US-2007/0191314.-   [58] Suli et al. (2004) Vaccine 22(25-26):3464-9.-   [59] WO95/11700.-   [60] U.S. Pat. No. 6,080,725.-   [61] WO2005/097181.-   [62] WO2006/113373.-   [63] Potter & Oxford (1979) Br Med Bull 35: 69-75.-   [64] Greenbaum et al. (2004) Vaccine 22:2566-77.-   [65] Zurbriggen et al. (2003) Expert Rev Vaccines 2:295-304.-   [66] Piascik (2003) J Am Pharm Assoc (Wash D.C.). 43:728-30.-   [67] Mann et al. (2004) Vaccine 22:2425-9.-   [68] Halperin et al. (1979) Am J Public Health 69:1247-50.-   [69] Herbert et al. (1979) J Infect Dis 140:234-8.-   [70] Chen et al. (2003) Vaccine 21:2830-6.-   [71] Current Protocols in Molecular Biology (F. M. Ausubel et al.,    eds., 1987) Supplement 30.-   [72] Smith & Waterman (1981) Adv. Appl. Math. 2: 482-489.

The invention claimed is:
 1. A reassortant influenza A virus comprisingan HA segment, an NA segment and backbone segments PA, PB1, PB2, NP, NSand M, wherein the backbone segments are from two donor strains, whereinthe PB1 segment is from the A/California/07/09 influenza strain and allother backbone segments are from the PR8-X influenza strain, furtherwherein the PA segment has the sequence of SEQ ID NO: 1, the PB2 segmenthas the sequence of SEQ ID NO: 3, the NP segment has the sequence of SEQID NO: 4, the M segment has the sequence of SEQ ID NO: 5, and the NSsegment has the sequence of SEQ ID NO:
 6. 2. The reassortant influenza Avirus of claim 1, wherein the PB1 segment has at least 95%, at least 99%identity, or 100% identity with the sequence of SEQ ID NO:
 16. 3. Thereassortant influenza A virus of claim 1, wherein the HA segment is froman H1 influenza strain.
 4. A reassortant influenza A virus comprising anHA segment, an NA segment and backbone segments PA, PB1, PB2, NP, NS andM, wherein the backbone segments are from two donor strains, furtherwherein at least one backbone segment is from the A/California/07/09influenza strain and: a) the PB2 segment comprises a lysine in theposition corresponding to amino acid 389 of SEQ ID NO: 3 when aligned toSEQ ID NO: 3, using a pairwise alignment algorithm; b) the PB2 segmentcomprises an asparagine in the position corresponding to amino acid 559of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using a pairwise alignmentalgorithm; c) the PA genome segment comprises a lysine in the positioncorresponding to amino acid 327 of SEQ ID NO: 1 when aligned to SEQ IDNO: 1, using a pairwise alignment algorithm; d) the PA segment comprisesan aspartic acid in the position corresponding to amino acid 444 of SEQID NO: 1 when aligned to SEQ ID NO: 1, using a pairwise alignmentalgorithm; e) the PA segment comprises an aspartic acid in the positioncorresponding to amino acid 675 of SEQ ID NO: 1 when aligned to SEQ IDNO: 1, using a pairwise alignment algorithm; f) the NP segment comprisesa threonine in the position corresponding to amino acid 27 of SEQ ID NO:4 when aligned to SEQ ID NO: 4 using a pairwise alignment algorithm; org) the NP segment comprises an asparagine in the position correspondingto amino acid 375 of SEQ ID NO: 4 when aligned to SEQ ID NO: 4, using apairwise alignment algorithm.
 5. The reassortant influenza A strain ofclaim 4, wherein: a) the PB2 segment comprises a lysine in the positioncorresponding to amino acid 389 of SEQ ID NO: 3 and asparagine in theposition corresponding to amino acid 559 of SEQ ID NO: 3 when aligned toSEQ ID NO: 3, using a pairwise alignment algorithm; b) the PA segmentcomprises a lysine in the position corresponding to amino acid 327;aspartic acid in the position corresponding to amino acid 444 of SEQ IDNO: 1 and aspartic acid in the position corresponding to amino acid 675when aligned to SEQ ID NO: 1, using a pairwise alignment algorithm; c)the NP genome segment comprises a threonine in the positioncorresponding to amino acid 27 of SEQ ID NO: 4 and asparagine in theposition corresponding to amino acid 375 when aligned to SEQ ID NO: 4,using a pairwise alignment algorithm; or d) the influenza A strain is aH1 strain.
 6. The reassortant influenza A strain of claim 5, wherein thePB2 segment comprises a lysine in the position corresponding to aminoacid 389 of SEQ ID NO: 3 and asparagine in the position corresponding toamino acid 559 of SEQ ID NO: 3 when aligned to SEQ ID NO: 3, using apairwise alignment algorithm, the PA genome segment comprises a lysinein the position corresponding to amino acid 327; aspartic acid in theposition corresponding to amino acid 444 of SEQ ID NO: 1 and asparticacid in the position corresponding to amino acid 675 when aligned to SEQID NO: 1, using a pairwise alignment algorithm, and the NP genomesegment comprises a threonine in the position corresponding to aminoacid 27 of SEQ ID NO: 4 and asparagine in the position corresponding toamino acid 375 when aligned to SEQ ID NO: 4, using a pairwise alignmentalgorithm.